Kooperativer Bibliotheksverbund

In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 230-230

Abstract: Rapid advancements in cancer genomics and in the development of targeted therapies provide expanding opportunities to use genomic profiling to improve patient outcomes. However, most patients do not have access to clinical genomic profiling platforms, and currently available assays capture a small set of known mutations or translocations tailored to specific tumor types. The spectrum of somatic alterations in leukemia, lymphoma, and myeloma includes substitutions, insertions/deletions (indels), copy number alterations (CNAs) and gene fusions; no current assay captures the different types of alterations in a single clinical genomic test. We developed a novel, CLIA-certified next-generation sequencing-based assay designed to provide targeted assessment of the genomic landscape of hematologic malignancies, including identification of all classes of genomic alterations using archived FFPE, blood and bone marrow aspirate samples with high accuracy in a clinically relevant timeframe. Methods DNA and RNA were successfully extracted from 350/362 (96%) specimens from 319 patients, including 57 FFPE samples, 150 blood samples and 142 bone marrow aspirates. The initial sample cohort included 20 ALL, 83 AML, 53 CLL, 57 DLBCL, 48 MDS, 32 MPN and 57 multiple myeloma samples. Adaptor ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 374 cancer-related genes and 24 frequently rearranged genes by DNA-seq, and 258 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine), averaging 590x for DNA and 〉 20M total pairs for RNA, to enable the sensitive and specific detection of substitutions, indels, CNAs and gene fusions. Results Sufficient tumor content (≥20%) was present in 317/350 (91%) of the samples (289/319 patients), and a total of 885 alterations were identified (3.1 alterations per sample), including 555 base substitutions, 213 indels, 36 splice mutations, 51 CNAs and 36 fusions/rearrangements. The most frequent alterations across all hematologic malignancies included mutations in TP53 (9%), ASXL1, KRAS, NRAS, IDH2, TET2, SF3B1, JAK2, MLL2, DNMT3A, RUNX1, and SRSF2 (2-5% each); FLT3 ITDs (2%); MLL PTDs (1%); homozygous loss of CDKN2A/B (3%); and focal amplification of REL (1%). Rearrangements in BCL2/6, MYC, MLL, MLL2, NOTCH2, ABL1 and ETV6 were identified using DNA and RNA targeted sequencing, demonstrating the ability of this platform to reliably identify gene fusions with immediate clinical relevance. Overall high accuracy of the assay for substitutions, indels and CNAs was previously demonstrated by extensive validation studies achieving 95-99% across alteration types with high specificity (PPV 〉 99%) [Frampton et al, Nat Biotech, in press]. Comparison of detected alterations to previous molecular testing for JAK2, NPM1, IDH2, FLT3 and CEBPA in MPN/AML samples demonstrated 97% sensitivity (33/34) in our ability to identify known mutations in these clinical samples. We identified additional clinically relevant mutations that were not detected using standard clinical assays, including alterations in JAK2, FLT3 and IDH2, which can inform therapeutic decisions. The use of our content rich sequencing platform allowed us to identify clinically actionable mutations in hematologic malignancies, including IDH1/2 mutations in a spectrum of myeloid/lymphoid malignancies, recurrent BRAF mutations in refractory CLL and myeloma, and mutations in the JAK-STAT signaling pathway in diffuse-large B cell lymphoma. These results demonstrate that a targeted sequencing platform which includes a large set of known disease alleles/therapeutic targets can identify mutations with therapeutic relevance in disease contexts where gene-specific assays are not currently performed in the clinical setting. Conclusions We have developed a sensitive, high throughput assay to detect somatic alterations in hundreds of genes known to be deregulated in hematologic malignancies, which can be used for clinical sequencing of frozen/paraffin samples. We demonstrate that targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations in genes known to be therapeutic targets in a broad spectrum of hematologic malignancies. Disclosures: Lipson: Foundation Medicine, Inc: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc: Employment, Equity Ownership. Otto:Foundation Medicine, Inc: Employment, Equity Ownership. Yelensky:Foundation Medicine, Inc: Employment, Equity Ownership. Wang:Foundation Medicine, Inc: Employment, Equity Ownership. He:Foundation Medicine, Inc: Employment, Equity Ownership. Rampal:Foundation Medicine: Consultancy. Brennan:Foundation Medicine, Inc: Employment, Equity Ownership. Brennan:Foundation Medicine, Inc: Employment, Equity Ownership. Young:Foundation Medicine, Inc: Employment, Equity Ownership. Donahue:Foundation Medicine, Inc: Employment, Equity Ownership. Sanford:Foundation Medicine, Inc: Employment, Equity Ownership. Greenbowe:Foundation Medicine, Inc: Employment, Equity Ownership. Frampton:Foundation Medicine, Inc: Employment, Equity Ownership. Fichtenholtz:Foundation Medicine, Inc: Employment, Equity Ownership. Young:Foundation Medicine, Inc: Employment, Equity Ownership. Erlich:Foundation Medicine, Inc: Employment, Equity Ownership. Parker:Foundation Medicine, Inc: Employment, Equity Ownership. Ross:Foundation Medicine, Inc: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment, Equity Ownership. Levine:Foundation Medicine, Inc: Consultancy.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V122.21.230.230

Language: English

Publisher: American Society of Hematology

Publication Date: 2013

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 127, No. 24 ( 2016-06-16), p. 3004-3014

Abstract: Novel clinically available comprehensive genomic profiling of both DNA and RNA in hematologic malignancies. Profiling of 3696 clinical hematologic tumors identified somatic alterations that impact diagnosis, prognosis, and therapeutic selection.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood-2015-08-664649

Language: English

Publisher: American Society of Hematology

Publication Date: 2016

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 1761-1761

Abstract: Whole genome and exome sequencing studies have identified numerous genomic alterations in DLBCL, but these methods have limited applicability for the clinical care of lymphoma patients due to cost, specific tissue requirements, and laborious bioinformatic analysis. FoundationOne-Heme (FOH) is a novel next-generation sequencing platform designed to provide targeted assessment of the genomic landscape of hematologic malignancies, including identification of mutations within specific genes, copy number changes, and translocations. FOH can be performed on small quantities of formalin-fixed paraffin-embedded (FFPE) tissue, detect rare variants due to extensive depth of sequencing coverage, and rapidly provide results via streamlined bioinformatic interpretation. Here we report the first experience using this novel platform to evaluate the genetic landscape of DLBCL. Genomic DNA and total RNA were isolated from FFPE tissue on a cohort of 53 cases of DLBCL, including de novo (n=30), relapsed/refractory (n=12), and large cell transformation from low-grade lymphoma (n=11). The cohort included 25 cases with combined MYC and BCL2 overexpression by IHC (criteria for positivity: 〉 40% MYC, 〉 70% BCL2), of which only one had a known translocation involving MYC. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom bait sets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 frequently-rearranged genes for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 〉 658x for DNA and 〉 20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. Significant non-synonomous variants were identified as mutations from the COSMIC database, amplifications of established oncogenes, or homozygous deletions and/or clear loss-of-function mutations of known tumor suppressors. The DNA sequencing component of FOH detected translocations in BCL2, BCL6, and MYC, while the RNA sequencing component detected fusion transcripts involving BLC6 and MYC, in agreement with independent cytogenetic analysis via karyotype and FISH where available. The assay detected copy number alterations of 44 different genes, most commonly amplification of REL (15%) or loss of CDKN2A/CDKN2B (17%). The most frequent alterations of known significance are detailed in Figure 1. The most commonly altered gene was CDKN2A, exhibiting either homozygous deletion or loss of function mutation in 28% of cases. Chromatin modifying factors (e.g. MLL2, CREBBP, EZH2) represented the most frequently altered biologic category with alterations occurring in 〉 50% of cases. Recurrent alterations in components of the Notch pathway (NOTCH1/2/4, FBXW7, SPEN), each predicted to activate the pathway, were identified in 23% of cases. Cell-of-origin was determined as per the Hans model using IHC for CD10, BCL6, and IRF4/MUM1; CD79B mutations were detected exclusively in non-GCB and EZH2 mutations were found exclusively in GCB-phenotype cases. Furthermore, IHC MYC+/BCL2+ de novo DLBCL cases (n=11) exhibited more frequent hypermutation of PIM1 (46%) compared with the 19 cases of IHC MYC-/BCL2- de novo DLBCL (11%). When comparing the various clinical categories, we found that mutations in tumor suppressors were significantly more common in relapsed/refractory than de novo DLBCL (47% vs 75%, p=0.02). Alterations in TP53 were most frequently observed in transformed lymphoma (55%). Our results demonstrate the feasibility of using a targeted next-generation sequencing platform on FFPE clinical specimens from patients with DLBCL as a means of providing an integrated analysis of gene mutations, copy number alterations, and translocations. This streamlined approach combines multiple molecular and cytogenetic tests into a single platform and uses a small amount of tissue to perform a multifaceted assessment of genomic alterations with potential diagnostic, prognostic, and therapeutic implications. Future efforts will be directed at analyzing additional cases of DLBCL to better establish the biologic and clinical significance of the observed genetic alterations, and to prospectively incorporate this novel platform to select patients for mechanism-based targeted therapy. Disclosures: Intlekofer: Foundation Medicine, Inc: Consultancy. Levine:Foundation Medicine, Inc: Consultancy. Zelenetz:Foundation Medicine, Inc: Consultancy. Palomba:Foundation Medicine, Inc: Consultancy. van den Brink:Foundation Medicine, Inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Young:Foundation Medicine, Inc: Employment. He:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Younes:Foundation Medicine, Inc: Consultancy.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V122.21.1761.1761

Language: English

Publisher: American Society of Hematology

Publication Date: 2013

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 1072-1072

Abstract: Background: The clinical utility of mutation profiling in hematologic malignancies requires robust detection of all classes of genomic alterations in a single analysis across different tumor types. We developed a novel, hybrid capture-based NGS assay (FoundationOne® Heme) designed to comprehensively assess the genomic landscape of hematologic malignancies from archived FFPE, blood and bone marrow aspirate samples, sequencing both DNA and RNA to improve sensitivity for driver fusion events. We have analyzed the genomic alterations including substitutions, indels, copy number alterations and genomic rearrangements of 795 samples with various hematolymphoid malignancies including lymphoma, leukemia and multiple myeloma to demonstrate the diagnostic, therapeutic and prognostic utility of this test in routine clinical practice. Method: Genomic profiles of 746 out of 795 (94%) consecutive samples received in a CLIA-certified laboratory (Foundation Medicine) were successfully characterized by the FoundationOne® Heme test including 527 from bone marrow aspirates, 176 FFPE samples and 92 samples derived from blood. The clinical cohort is composed of 375 multiple myeloma cases, 185 leukemia cases, 150 lymphoma cases, 75 MDS/MPN cases, and 15 unknown hematologic disorder cases (Figure A). Adaptor-ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 405 blood cancer-related genes and 31 frequently rearranged genes by DNA-seq, and 265 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 498x for DNA and ~7M on-target unique pairs for RNA, to enable the sensitive and specific detection of substitutions, indels, copy number alteration and gene rearrangements. Results: 705/746 (95%) patients had at least one alteration reported as a somatic driver mutation, 68% of cases harbored at least one alteration linked to a targeted therapy or would inform enrollment in a clinical trial consistent with therapeutic relevance. These potentially actionable alterations included mutations in KRAS (14% of cases), NRAS (13%), CDKN2A (8%), DNMT3A (6%), IDH1/2 (5%), BRAF (4%) and FLT3 (3%) (Figure B). In addition, 64% of cases harbored at least one alteration that have been shown to predict outcome and have prognostic relevance, including TP53 (14% of cases), TET2 (7%), ASXL1 (7%), CDKN2B (5%), CREBBP (5%), MLL (5%) and NPM1 (3%)(Figure B). We also detected 344 genomic rearrangements in 280/746 (38%) patients. Of the 344 reported rearrangements, 166 were known fusions in various disease types and 178 were novel rearrangements involving known oncogenes and tumor suppressor genes, including a novel RCSD1-ABL2 fusion in a patient with B-cell ALL who will derive benefit from kinase inhibitor therapy as part of their anti-leukemic regimen. Genomic rearrangements (detected in 38% of cases) can lead to various functional consequences: 260 (76%) rearrangements detected in this cohort are predicted to create activating fusions; 19 (6%) rearrangements are predicted to be activating intragenic rearrangements in MLL, FLT3 and CARD11; and 60 (17%) rearrangements are likely truncating events in tumor suppressors. Conclusions: We demonstrated that hybrid capture-based targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations in genes known to be therapeutic targets or prognostic predictors in a broad spectrum of hematologic malignancies. Moreover, our data suggests that the FoundationOne® Heme comprehensive genomic profiling test can alter therapeutic strategy in routine clinical practice. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures He: Foundation Medicine: Employment, Equity Ownership. Nahas:Foundation Medicine, Inc: Employment. Wang:Foundation Medicine, Inc.: Employment, Equity Ownership. Young:Foundation Medicine: Employment, Equity Ownership. Brennan:Foundation Medicine, Inc: Employment. Donahue:Foundation Medicine: Employment. Young:Foundation Medicine, Inc: Employment. Ross:Foundation Medicine, Inc.: Employment, Equity Ownership. Morosini:Foundation Medicine, Inc. : Employment, Equity Ownership. van den Brink:Foundation Medicine, Inc: Consultancy. Intlekofer:Foundation Medicine, Inc: Consultancy. Dogan:Foundation Medicine, Inc: Consultancy. Armstrong:Epizyme, Inc: Consultancy. Yelensky:Foundation Medicine, Inc.: Employment, Equity Ownership. Otto:Foundation Medicine, Inc.: Employment. Lipson:Foundation Medicine, Inc.: Employment, Equity Ownership. Stephens:Foundation Medicine, Inc.: Employment, Equity Ownership. Miller:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V124.21.1072.1072

Language: English

Publisher: American Society of Hematology

Publication Date: 2014

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 704-704

Abstract: Background: A variety of next-generation sequencing methods have been used to investigate the genomic landscape of primary lymphoma patient samples, including whole-genome, whole-exome, and RNA sequencing. In diffuse large B cell lymphoma (DLBCL), sequencing studies identified numerous genomic alterations (GAs) of potential clinical relevance, but the distribution and frequency of GAs have not been precisely determined. Discrepancies in existing data likely arise from variability in types of specimens examined, sequencing technologies employed, and depth of coverage utilized to identify GAs. In this study, we performed comprehensive DNA/RNA sequencing of genes known to be important across the spectrum of hematologic malignancies in order to determine the nature and prevalence of GAs with potential diagnostic, prognostic, or therapeutic implications in a cohort of 112 well-annotated clinical DLBCL cases. Methods: We performed hybridization capture of 405 cancer-related genes and 31 genes commonly rearranged in cancer (FoundationOne Heme) and 265 frequently rearranged genes for RNA-seq applied to ³50ng of DNA and sequenced to high, uniform coverage. Genomic alterations, including short variants (small indels and base substitutions), rearrangements, and copy number alterations, were determined. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 〉 500x for DNA and 〉 20,000,000 total pairs for RNA, to enable the sensitive and specific detection of GAs. Significant non-synonomous variants were identified as mutations from the COSMIC database, amplifications of established oncogenes, or homozygous deletions and/or clear loss-of-function mutations of known tumor suppressors. Results: GAs with diagnostic, prognostic, or therapeutic relevance were identified in 96% of cases, with a median of 5 (range 0 to 12) alterations per sample. Figure 1 shows the frequencies of different GAs. De novo DLBCL tended to exhibit fewer GAs (median 4) than relapsed/refractory (median 6) or transformed disease (median 6) (p=0.179; Wilcoxon rank sum). Compared with reported frequencies ranging from 5 to 44%, we detected alterations in CREBBP/EP300 in 21% of cases, with CREBBP mutations preferentially found in germinal-center B cell-like (GCB) compared to non-GCB DLBCL (26% vs. 8%; p=0.02; Pearson's chi-squared). Although previously described only in Burkitt lymphoma, we identified mutations in ID3 (L70P, P98R, Q100P) and TCF3 (N551K) in GCB-phenotype DLBCL cases with aggressive pathologic features and no MYC expression by IHC. Additional novel findings included alterations of genes involved in cellular metabolism in 18% of cases, including one IDH2 R172M mutation and two cases with SDHA L649fs* truncating mutations. We also identified several mutations more commonly found in solid tumors and leukemias, including BRAF V600E and KRAS G13D. With respect to genomic rearrangements, combined DNA/RNA capture and sequencing detected translocations/fusions in MYC, BCL2, and BCL6, which were concordant with cytogenetics/FISH analysis. We also identified rearrangements involving TBXL1XR1-P63, NOTCH2, SOCS1, and ETV6. Copy number alterations were detected in 26 different genes, including amplifications of CD274/PDCD1LG2 (PD-L1/PD-L2) (n=4) that were restricted to non-GCB cases. Alterations in TP53 were more frequently observed in transformed (42%) and relapsed/refractory (26%) compared to de novo DLBCL (12%; p=0.007; Pearson's chi-squared). TP53 mutations predicted for lack of response to chemotherapy, with chemo-refractory disease occurring in 48% of TP53-mutant patients compared to 10% of patients with intact TP53 (p=0.0004; Fisher's exact). Truncating mutations or deletions of RB1, albeit rare (5% of cases), were the most significant negative prognostic factor and were associated with therapeutic resistance and poor overall survival. Conclusions: Our results demonstrate the utility of comprehensive combined DNA/RNA next-generation sequencing as a promising method to identify clinically relevant GAs in clinical lymphoma specimens. This streamlined approach has the potential to combine multiple molecular and cytogenetic tests into a single platform. Future efforts will be directed at incorporating this approach both retrospectively and prospectively into clinical trials to identify predictive biomarkers to guide therapeutic decisions. Figure 1 Figure 1. Disclosures Intlekofer: Foundation Medicine, Inc: Consultancy. Levine:Foundation Medicine, Inc: Consultancy. Zelenetz:Foundation Medicine, Inc: Consultancy. Dogan:Foundation Medicine, Inc: Consultancy. Palomba:Foundation Medicine, Inc: Consultancy. van den Brink:Foundation Medicine, Inc: Consultancy. Brennan:Foundation Medicine, Inc: Employment. Young:Foundation Medicine, Inc: Employment. He:Foundation Medicine: Employment. Nahas:Foundation Medicine, Inc: Employment. Yelensky:Foundation Medicine, Inc: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Miller:Foundation Medicine, Inc: Employment. Younes:Foundation Medicine, Inc: Consultancy.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V124.21.704.704

Language: English

Publisher: American Society of Hematology

Publication Date: 2014

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Journalism & Mass Communication Educator, SAGE Publications, Vol. 77, No. 4 ( 2022-12), p. 393-413

Abstract: The American news media is facing a crisis of public trust fueled, in part, by an epistemological disagreement of what constitutes quality news. Through a series of 50 interviews with journalism professionals and students, this research investigates how evidence-based journalism is created by gathering data on expert and novice research practices. Interviews were transcribed, coded, and analyzed through the lens of the ACRL Framework to provide the foundation for a journalism information literacy framework. This analysis can be used to inform library instruction and higher education journalism curricula and offers insights and greater transparency on the processes that underlie trustworthy news.

Type of Medium: Online Resource

ISSN: 1077-6958 , 2161-4326

URL: Article

DOI: 10.1177/10776958221117050

Language: English

Publisher: SAGE Publications

Publication Date: 2022

detail.hit.zdb_id: 2070252-8

SSG: 3,5

In: Cancer Research, American Association for Cancer Research (AACR), Vol. 74, No. 19_Supplement ( 2014-10-01), p. 3570-3570

Abstract: Background: Next-generation sequencing (NGS) is rapidly becoming an indispensable cancer diagnostic, as it can detect most genomic alterations in a single assay from limited tissue. We developed a novel, NGS-based assay designed to provide targeted assessment of the genomic landscape of hematologic malignancies from archived FFPE, blood and bone marrow aspirate samples, sequencing both DNA and RNA to improve sensitivity for driver fusion events which are common in these tumors. Methods: The high accuracy of the assay for detection of substitutions, indels and CNAs was previously demonstrated by extensive validation studies achieving 95-99% across all alteration types with high specificity (PPV & gt;99%) [Frampton et al, Nat Biotech, 2013]. To validate assay performance in detecting gene fusions we created reference samples by mixing 21 cell-lines with previously characterized fusions in 39 combinations representing 167 fusion events in 10-50% tumor cell fractions. In addition, we confirmed accuracy in 76 clinical hematologic FFPE and bone-marrow samples profiled for 212 substitutions, indels and fusions in 11 genes by Sanger sequencing, PCR, fragment sizing and FISH. DNA and RNA were extracted from all samples; adaptor ligated sequencing libraries were captured by solution hybridization using custom baits targeting 405 cancer related genes by DNA-seq, and 265 frequently rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq) in a CLIA-certified laboratory (Foundation Medicine), averaging 467x for DNA and 6M unique pairs for RNA. Results: On reference samples, sensitivity for detection of fusions events reached & gt;99% for tumor cell fractions of 20-50%, and 97% for tumor fraction of 10%, all with high specificity (PPV & gt;95%). Robust performance translated to the clinical samples: we observed a concordance rate of 98.6% relative to prior calls, with only 3/212 differing calls (2+, 1-) by NGS. 129 additional known oncogenic alterations in 57 different genes were detected in these samples, for a total of 3.1 alterations per sample. Analysis of 290 additional leukemia, lymphoma and myeloma patient samples revealed known and novel gene fusions in 15% of cases, more than half of which were identifiable only by RNA-seq. Conclusions: We describe the analytic validation of a sensitive, high throughput assay to detect somatic alterations in hundreds of genes known to be deregulated in hematologic malignancies, which can be used to identify a spectrum of somatic alterations from blood, bone marrow and paraffin embedded patient samples. We demonstrate that targeted DNA and RNA sequencing can be used to identify all classes of genomic alterations, including gene fusions, with high accuracy. This approach offers the opportunity to streamline the characterization of genomic alterations in hematologic malignancies and to expand targeted treatment options for patients with liquid tumors. Citation Format: Doron Lipson, Michelle K. Nahas, Geoff A. Otto, Jie He, Kai Wang, Kristina M. Knapp, Kristina W. Brennan, Amy L. Donahue, Lauren E. Young, Geneva Young, Alex Fichtenholtz, Jeffrey S. Ross, Roman Yelensky, Philip J. Stephens, Vincent A. Miller, Ross Levine. Development and validation of a clinical next generation sequencing-based assay for hematologic malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3570. doi:10.1158/1538-7445.AM2014-3570

Type of Medium: Online Resource

ISSN: 0008-5472 , 1538-7445

URL: Article

DOI: 10.1158/1538-7445.AM2014-3570

Language: English

Publisher: American Association for Cancer Research (AACR)

Publication Date: 2014

detail.hit.zdb_id: 2036785-5

detail.hit.zdb_id: 1432-1

detail.hit.zdb_id: 410466-3

In: Circulation, Ovid Technologies (Wolters Kluwer Health), Vol. 144, No. 23 ( 2021-12-07), p. 1845-1855

Abstract: Despite advances in surgery and pharmacotherapy, there remains significant residual ischemic risk after coronary artery bypass grafting surgery. Methods: In REDUCE-IT (Reduction of Cardiovascular Events With Icosapent Ethyl–Intervention Trial), a multicenter, placebo-controlled, double-blind trial, statin-treated patients with controlled low-density lipoprotein cholesterol and mild to moderate hypertriglyceridemia were randomized to 4 g daily of icosapent ethyl or placebo. They experienced a 25% reduction in risk of a primary efficacy end point (composite of cardiovascular death, myocardial infarction, stroke, coronary revascularization, or hospitalization for unstable angina) and a 26% reduction in risk of a key secondary efficacy end point (composite of cardiovascular death, myocardial infarction, or stroke) when compared with placebo. The current analysis reports on the subgroup of patients from the trial with a history of coronary artery bypass grafting. Results: Of the 8179 patients randomized in REDUCE-IT, a total of 1837 (22.5%) had a history of coronary artery bypass grafting, with 897 patients randomized to icosapent ethyl and 940 to placebo. Baseline characteristics were similar between treatment groups. Randomization to icosapent ethyl was associated with a significant reduction in the primary end point (hazard ratio [HR], 0.76 [95% CI, 0.63–0.92] ; P =0.004), in the key secondary end point (HR, 0.69 [95% CI, 0.56–0.87]; P =0.001), and in total (first plus subsequent or recurrent) ischemic events (rate ratio, 0.64 [95% CI, 0.50–0.81]; P =0.0002) compared with placebo. This yielded an absolute risk reduction of 6.2% (95% CI, 2.3%–10.2%) in first events, with a number needed to treat of 16 (95% CI, 10–44) during a median follow-up time of 4.8 years. Safety findings were similar to the overall study: beyond an increased rate of atrial fibrillation/flutter requiring hospitalization for at least 24 hours (5.0% vs 3.1%; P =0.03) and a nonsignificant increase in bleeding, occurrences of adverse events were comparable between groups. Conclusions: In REDUCE-IT patients with a history of coronary artery bypass grafting, treatment with icosapent ethyl was associated with significant reductions in first and recurrent ischemic events. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01492361.

Type of Medium: Online Resource

ISSN: 0009-7322 , 1524-4539

URL: Article

DOI: 10.1161/CIRCULATIONAHA.121.056290

Language: English

Publisher: Ovid Technologies (Wolters Kluwer Health)

Publication Date: 2021

detail.hit.zdb_id: 1466401-X

In: Blood, American Society of Hematology, Vol. 124, No. 21 ( 2014-12-06), p. 286-286

Abstract: Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genomic lesions. However, given recent recognition that multiple genomically distinct sub-clones can exist in AML, there is a risk that there may be selection for sub-clones from the transplanted sample that might not fully represent the patient’s disease. We transplanted 160 (70 T-ALL 56 AML, 32 B-ALL, 2 MLL) patient samples of which 120 engrafted into at least 1 irradiated NSG mouse. 45 AML samples engrafted with a median latency 107+/-41 days. Transplantation of 6 PDX AML samples resulted in immunophenotypically identical disease within 87+/-35 days. 2 MLL samples engrafted in 100% of mice with a median latency of 103+/-13 days. 25 B-ALL samples engrafted with a median latency of 95+/-44 days. Secondary transplantation of 3 PDX B-ALL samples resulted in engraftment of leukemia cells with an identical immunophenotype in 100% of transplanted mice within 52+/-3 days. 48 T-ALL samples engrafted in at least one mouse within 50 days. Secondary transplantation of a single T-ALL PDX sample resulted in 100% engraftment within 31+/-10 days. Genomic DNA and total RNA were isolated from 150 (AML: 16Pt+33PDX; MLL 2Pt+6PDX; B-ALL 17Pt+38PDX; T-ALL 19Pt+19PDX) samples. Adaptor ligated sequencing libraries were captured by solution hybridization using baitsets for 405 cancer-related genes and selected introns for 31 genes frequently rearranged for DNA-seq, and 405 cancer-related and 265 genes frequently rearranged for RNA-seq. All libraries were sequenced averaging 〉 500x for DNA and 〉 6M total pairs for RNA (HiSeq). We detected on average 23+/-12 including a mean 5+/-4 known pathogenic variants such as CDKN2A/B deletion (20/13); FLT3 (SNV & -ITD) and NOTCH (11 ea); WT1 and TP53 (10 ea); NRAS (9); PTPN11 (7); NPM1c, PTEN, and KRAS (6) DNMT3A, IDH1/2, and ASXL1 (5 ea); FBXW7, CEBPA, and TET2 (4 ea); PHF6 and NF1 (3 ea); IKZF1, ATM, and JAK2 (2 ea). Analyses of fusion RNA molecules detected known fusions: MLL-AF4 (4); MLL-AF9 (2), CRLF2-P2RY8, ETV6-RUNX1 or TEL-AML1, PBX1-TCF3 (2 ea); MLL-AF10, MLL-ELL, MLL-EP300, MLL-PTD, BCR-ABL, BCL2-IGK, MYH11-CBFB, along with novel fusions: TCF3-OAZ1, RB1-RCBTB2, PAX5-FLI1, and PAX5-MSI2. The mutations found in the 54 patient samples were consistently identified in the 96 PDX, however some cases showing variation in allele frequency between diagnostic and engrafted samples. Collectively, all 1420 and 288 disease relevant variant allele frequency (VAF) correlated significantly between patient and PDX samples (R2=0.55, R2=0.43), respectively. We then assessed VAF changes from diagnostic to PDX sample as a measure of clonal concordance. Diagnostic and PDX sample were considered discordant if at least one disease relevant VAF demonstrated significant variation between these samples, accounted for small variability of infrequent variances considering SD of sequencing detection. 31 samples were scored as concordant and 23 as discordant which were similarly distributed between disease lineages and did not correlate with diseases status, future relapse or overall survival. Using the same rules we further accessed concordance only between PDX samples in 23 cases when patient samples were transplanted into multiple mice. All 10 groups of PDX samples that were concordant with patient samples were also concordant within the groups. 5 groups of PDX samples that were discordant with patient samples were concordant within groups. 8 groups of PDX samples that were discordant with patient samples were also discordant within their groups. Overall 15 samples produced concordant engraftment in mice and 8 samples produced discordant engraftment. We hypothesized that specific genomic lesions in the 8 groups might underline this discordance. Mutations of FLT3, RAS, TP53, PTPN11 and NOTCH1 correlated with clonal discordance. These findings show that the leukemias that are engrafted in mice mirror the genomic diversity of primary leukemia samples, and that the majority of PDX samples have a genotype similar to that observed in the clinical isolate. More importantly, our data demonstrate the feasibility of developing a large, genetically annotated bank of PDX leukemia models that can be used to test and credential novel therapeutics that target driver mutations in different leukemia subsets. Disclosures Stein: Seattle Genetics, Inc.: Research Funding; Janssen Pharmaceuticals: Consultancy. Wang:Foundation Medicine Inc: Employment. Miller:Foundation Medicine: Employment. Armstrong:Epizyme: Consultancy.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V124.21.286.286

Language: English

Publisher: American Society of Hematology

Publication Date: 2014

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7

In: Blood, American Society of Hematology, Vol. 122, No. 21 ( 2013-11-15), p. 1328-1328

Abstract: Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genetic lesions. However, given our recent recognition that multiple genetically distinct subclones can exist in AML, there is a risk that there may be selection for sub-clones from the xenotransplanted sample that might not fully represent the patient’s disease. We sought to establish a collection of genetically defined AML samples capable of engraftment in immunodeficient mice. We transplanted 30 AML patient samples; within 150 days (median 91 days) post transplantation 12 samples produced human CD45+ CD33+ CD19- CD3- engraftment in one or multiple NSG mice. Median patient sample amplification in 25 mice was 21 fold. Genomic DNA and total RNA was isolated from 7 AML patient samples (3 diagnostic samples from patients who remain in remission; 2 diagnostic samples from patients who later relapsed, 2 diagnostic samples from patients with refractory disease) and 14 matched xenotransplanted samples (2 mice per patient sample). Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 〉 499x for DNA and 〉 20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. The mutations found in the 7 diagnostic samples were consistently identified in the 14 engrafted AML samples, but with some cases showing variation in allele frequency between diagnostic and engrafted samples. This finding shows that the human disease that engrafted in mice mimics the genetic makeup of the disease found in patients. We then assessed for allele frequency (AF) changes from diagnostic to xenografted sample as a measure of clonal progression. Clonal progression was defined as emergence of a clone carrying a novel genetic variant in the xenografted sample as compared to the diagnostic patient sample. Five patient samples (from 10 mice) did not show emergence of novel genetic lesions. In this group 2 patients had refractory disease and 3 patients remain in remission. Two patient samples (from 4 mice) demonstrated apparent emergence of novel genetic lesions not detected in diagnostic patient samples. Both of these patients have relapsed since the diagnostic samples were acquired. In the first case, both xenotransplanted mice engrafted with disease carrying NRAS N12S mutation (AF 0.05 and 0.09), which subsequent evaluation revealed to be present below the limit-of-detection (AF 0.004) in the clinical isolate obtained from patient presentation. We are currently conducting the same analysis on the relapsed sample from this patient. In the second case, both mice engrafted with disease carrying PTPN11 E76V (AF 0.03 and 0.0016) while the patient diagnostic sample did not contain any evidence of the alteration at 718x unique sequence coverage. Of note, one xenografted sample had an IDH1 R132C and another had IDH2 R140Q mutation, both of which have previously been shown to play a role in AML pathogenesis. Available AML cell lines do not carry IDH1/2 mutations, making it challenging to test IDH1/2 inhibitors in pre-clinical settings. These xenografted samples offer an opportunity to test such inhibitors. Overall we conclude that the xenotransplanted samples possess the diversity of genetic abnormalities found in diagnostic AML samples and thus can be used to assess efficacy of novel targeted therapies. We would like to further investigate a model in which the absence of clonal progression in xenografted samples would predict a better patient outcome, while emergence of novel clones might indicate an increased potential for relapse. We are currently expanding the study to include more diagnostic, xenotransplanted and relapsed samples to assess the associations between the ability of a sample to engraft in mice with clinical outcome and genetic/epigenetic lesions. Disclosures: Armstrong: Epizyme Inc.: Has consulted for Epizyme Inc. Other.

Type of Medium: Online Resource

ISSN: 0006-4971 , 1528-0020

URL: Article

DOI: 10.1182/blood.V122.21.1328.1328

Language: English

Publisher: American Society of Hematology

Publication Date: 2013

detail.hit.zdb_id: 1468538-3

detail.hit.zdb_id: 80069-7